STERILIZATI

ON
 Definition
• Sterilization: Process by which all living microorganisms including
viable spores, are either destroyed or removed from an article,
body surface or medium.

• Disinfection- Process that destroys or removes most if not all

pathogenic organisms but not bacterial spores.
 Definition
• Asepsis- Process where the chemical agents (called antiseptics)
applied to body surfaces (skin) will kill or inhibit the pathogenic
microorganisms (and also commensals) present on skin.

• Decontamination (or sanitization)- Reduction of pathogenic

microbial population to a level at which items are considered as
safe to handle without protective attire.
Factors influencing efficacy of sterilant / disinfectant

1. Organism load: Larger microbial population requires a

longer time to be destroyed.
2. Nature of organisms
Factors influencing efficacy of sterilant / disinfectant

3. Concentration of the chemical agent or the temperature of heat sterilization.

4. Nature of the sterilant /disinfectant
5. Duration of exposure
6. Temperature
Factors influencing efficacy of sterilant/ disinfectant

7. Local environment
o Heat kills more readily at an acidic pH.
o Biofilm are also a good example that prevents the entry of disinfectants to act on the
microorganisms that are embedded inside the biofilm.
Classification of sterilization methods
1.Sunlight 2.Drying
3.Heat
Dry heat- Flaming, Incineration, Hot air oven
Moist heat-
 Temperature below 1000C- pasteurization, water bath and inspissation
 Temperature at 1000C- e.g. boiling, steaming and tyndallisation
 Temperature above 1000C- e.g. autoclave
4.Filtration – depth filters and membrane filters
5.Radiation
Ionizing radiation-γ rays, X-rays and cosmic rays
Non-ionizing radiation- Ultraviolet (UV) and infrared rays
6. Ultrasonic vibration
PHYSICAL AGENTS OF STERILIZATION/
DISINFECTION

Method Principle

Sunlight Active microbicidal effect due to its content of ultraviolet rays.

Drying 70-80% of the weight of the bacterial cell is due to water. Drying,
therefore has a deleterious effect on many bacteria.

Dry heat Kills the organisms by charring, denaturation of bacterial protein,

oxidative damage and by the toxic effect of elevated levels of
electrolytes.

Moist heat Kills the microorganisms by denaturation and coagulation of proteins.

 Dry heat sterilization
Flaming Items are held in the flame of a Bunsen burner either for
long time or short time.
o Longer time exposure - for inoculating wires or loops, tips of forceps.
o For shorter period - mouth of test tubes.

Incineration Used for the disposal of waste materials.

o It burns (sterilizes) the anatomical waste and microbiology waste by
providing a very high temperature 870 to 1,200°C - converting the
waste into ash, flue gas, and heat.
 Hot air oven (Dry heat Sterilizer)

• Most widely used method of sterilization by

dry heat.
• It is electrically heated and is fitted with a fan
to ensure adequate and even distribution of
hot air in the chamber.
• It is also fitted with a thermostat which
maintains the chamber air at a chosen
temperature.
• Holding temperature and time of 160°C for 2
hours.
 Hot air oven (Dry heat Sterilizer)

• Materials sterilized:
o Glassware - syringes, petri dishes, flasks, pipettes and test tubes.
o Surgical instruments - scalpels, forceps, etc.
o Chemicals such as liquid paraffin, fats, glycerol, oil, and glove
powder, etc.
Hot air oven - precautions

• Overloading of hot air oven should be avoided.

• Material should be arranged in a manner so that free circulation of air is
maintained.
• Material to be sterilized should be dried completely.
• Cotton plugs should be used to close the mouths of test tubes, flasks, etc.
• Paper wrapping of the items should be done.
• Any inflammable material like rubber (except silicone rubber) should not be
kept inside the oven.
• Oven must be allowed to cool for two hours before opening the doors,
since the glassware may crack by sudden cooling.
 Moist Heat sterilization -
Below 100°C

Pasteurization • Method used for control of microorganisms from beverages like

fruit and vegetable, juices, beer, and dairy products, such as milk.
o Holder method (63°C for 30 mins).
o Flash method (72°C for 20 seconds followed by rapid cooling to
13° or lower).
o All nonsporing pathogens, including mycobacteria, brucellae and
salmonellae are killed except Coxiella burnetii which being
relatively heat resistant may survive in holder method.
Moist Heat sterilization -
Below 100°C

Water bath
• Used for disinfection of serum, body fluids and vaccines.
• Bacterial vaccines are disinfected at 60°C for 1 hour.
• Serum or heat labile body fluids can be disinfected at 56°C
for one hour.
Moist Heat sterilization -
Below 100°C

Inspissation • Process of heating an article on 3 successive days at 80–85ฐC

(fractional for 30 minutes.
sterilization) • Working principle:
o first exposure - kills all the vegetative forms.
o Intervals between the heating the remaining spores
germinate into vegetative forms which are then killed on
subsequent heating.
• Uses: Sterilization egg-based medium – Lowenstein Jensen
medium and Dorset’s egg medium; serum-based media—
Loeffler’s serum slope.
 Moist Heat sterilization
Moist heat at a temperature at 100°C

Boiling • Boiling of the items in water for 15 minutes kills most of the
vegetative forms.
• But do not kill the spores - not suitable for sterilization of surgical
instruments.

Koch’s or • useful for those media which are decomposed at high

Arnold’s steam temperature of autoclave.
sterilizer • Articles exposed to steam (100°C) at atmospheric pressure for 90
minutes.
Most of the vegetative forms are killed by this method
except thermophiles and spores.
 Moist Heat sterilization
at 100°C

Tyndallisation • Involves steaming at 1000C for 20minutes for 3 consecutive days.

or intermittent • Principle is similar to that of inspissation, except that here, the
sterilization temperature provided is 1000C, instead of 800C.
(named after • Used for sterilization of gelatin and egg, serum or sugar
John Tyndall) containing media which are damaged at higher temperature of
autoclave.
 Moist Heat sterilization
above 100°C

• Principle of Autoclave:
o Autoclave functions similar to a pressure cooker and follows the general laws of gas.
o Water boils when its vapour pressure equals that of the surrounding atmosphere.
o When the atmospheric pressure is raised, the boiling temperature is also raised.
o At normal pressure, water boils at 100°C but when pressure inside a closed vessel
increases, the temperature at which water boils also increases.
 Components of Autoclave
• Autoclave comprises of three parts
 Pressure chamber
Lid
Electrical heater.
Horizontal autoclave
and Vertical autoclave
 Sterilization Conditions

• Autoclave can be set to provide higher temperatures by adjusting the pressure

provided to the vessel.
o 121⁰C for 15 minutes at pressure of 15 lbs psi.
o 126 ⁰ C for 10 minutes at pressure of 20 psi.
o 133 ⁰ C for 3 minutes at pressure of 30 psi.
Uses of Autoclave

• Surgical instruments
• Culture media
• Autoclavable plastic containers
• Plastic tubes and pipette tips
• Solutions and water
• Biohazardous waste
• Glassware (autoclave resistible).
 Precautions to be taken
• Autoclave should not be used for sterilizing waterproof materials
• Materials are loaded in such a way that it allows efficient steam penetration
(do not overfill the chamber)
• Material should not touch the sides or top of the chamber
• Clean items and the wastes should be autoclaved separately
Types of Autoclaves

• Gravity displacement type autoclave: most commonly used.

• Positive pressure displacement type autoclave
• Negative pressure (vacuum) displacement type.
 Sterilization control

• Biological indicator: Spores of Geobacillus stearothermophilus (formerly

called Bacillus stearothermophilus). Spores are killed in 12 minutes at 121°C
• Chemical indicators
o Class I - external pack control, (e.g. autoclave tape)
o Class II - equipment control (Bowie-Dick test)
o Class IV/V - internal pack control.
• Physical control: For example, digital displays on the equipment displaying
temperature, time and pressure.
Filtration
• Filtration is an excellent way to remove the microbial
population in solutions of heat-labile materials like vaccine,
antibiotics, toxin, serum and sugar solution.
• Types of Filters
1. Depth Filters.
2. Membrane Filters
 Depth Filters
• Depth filters are porous filters composed of random mats of
metallic, polymeric, or inorganic materials.
• Advantages - Retain a large mass of particles before becoming
clogged, flow rate of the fluid is high and low cost
• Disadvantages- Not suitable for filtration of solution containing
bacteria.
• Depth filters - candle filters (Berkefeld filters), unglazed
porcelain (Chamberlain filters),asbestos filters (Seitz and
Sterimat filters) and sintered glass filters
 Membrane Filters
• Most widely used filters for bacterial filtration.
• Porous; retain all the particles on the surface that are
smaller than their pore size.
• Composed of cellulose acetate, cellulose nitrate,
polycarbonate, polyvinylidene fluoride, or other synthetic
materials
• Pore size:
o 0.22 µm - Most commonly used (removes most of the bacteria,
allowing the viruses to pass through)
o 0.45 µm - Used to retain coliforms.
o 0.8 µm - Used to remove airborne microorganisms in clean
rooms.
Filtration of Liquid
• To sterilize sera, sugar and antibiotic solutions.
• Separation of toxins and bacteriophages from bacteria.
• To obtain bacteria free filtrates of clinical samples for virus
isolation
• Purification of water—e.g. testing of water samples for
Vibrio cholera or typhoid bacilli.
 Filtration of Air
• Simple e.g. - surgical masks (that allow air in but keep microorganisms
out).
• Two important air filters that are used in biological safety cabinets and
laminar airflow systems):
oHEPA filters (High-efficiency particulate air filters) removes 99.97%
of particles that have a size of 0.3 µm or more.
o ULPA filters (Ultra-low particulate/penetration) removes from the
air at least 99.999% of dust, pollen, mold, bacteria and any airborne
particles with a size of 0.12 µm or larger.
 Ionizing radiation (cold
sterilization)
• Include, X-rays, gamma rays (from Cobalt 60 source), and cosmic rays.
• Mechanism: It causes breakage of DNA without temperature rise.
• Uses
o Disposable rubber or plastic syringes, infusion sets and catheters.
o Catgut sutures, bone and tissue grafts and adhesive dressings as well as antibiotics
and hormones.
o Irradiation of food (permitted in some countries).
• Advantages of ionizing radiation:
o High penetrating power, Rapidity of action and Temperature is not raised
• Sterilization control: Efficacy of ionizing radiation is tested by using Bacillus pumilus.
 Non-ionizing Radiation
• Includes infrared and ultraviolet radiations.
• Lethal but do not penetrate glass, dirt films, water; hence their use is
restricted.
• Recommended dose - 250–300 nm wavelength of UV rays, for 30 minutes
• Uses – For disinfection of clean surfaces in operation theaters, laminar flow
hoods as well as for water treatment.
• Because UV radiation burns the skin and damages eyes, hence the area
should be closed and UV lamps must be switched off immediately after use.
Ultrasound (Ultrasonic) Waves

• High-frequency ultrasonic and sonic sound waves disrupt bacterial

cells.
• Not reliable, hence is not used now a days.
Thank you